Project Summary In the brain, key focal points for control of metabolic function and feeding behavior are melanocortin receptors MC3R and MC4R. These receptors respond to two ligands, alpha-melanocyte stimulating hormone (a-MSH) and the agouti-related protein (AgRP), which act in opposite ways to promote negative and positive energy balance, respectively. Recent research demonstrates the profound importance of AgRP releasing neurons in metabolism and body weight homeostasis. The goal of this program is to understand the molecular basis of AgRP action, thus enabling new strategies for treating diverse conditions linked to obesity and metabolic diseases. New findings from our lab are significantly reshaping our understanding of a-MSH and AgRP action. The current paradigm posits that these molecules act to stimulate or suppress production of the cAMP second messenger. However, using protein design, we demonstrated that AgRP mutations in segments outside of the MCR binding core exert a profound influence on long term feeding, while leaving receptor affinity and cAMP suppression completely unchanged relative to wild-type. Moreover, new collaborative results find that AgRP promotes the opening of inward rectifying potassium channels through a cAMP independent mechanism, an effect that is directly dependent on these peripheral AgRP segments. Aim 1 of this application will expand these studies by identifying how AgRP sequence and conformation drive potassium channel currents. This will be tested through protein design, NMR structure determination, as well as with a new human AgRP obesity-linked mutant identified by whole exome analysis. Aim 2 will examine how syndecan-3, a negatively charged, membrane bound proteoglycan, facilitates AgRP signaling. This will be accomplished with biophysical experiments and through comparison studies where designed proteins are administered to wild-type and syndecan-3 knockout mice, followed by feeding trials and brain tissue imaging. Aim 3 will expand on AgRP design efforts to produce stable proteins to test the role of proteolytic resistance in promoting long-term AgRP action, and as leads for treating cancer cachexia, a wasting condition characterized by extreme loss of appetite and lean tissue degradation.